Inhibiting T-cell proliferation

ABSTRACT

Enhancement of T-cell proliferation in a host inhibition is provided by administering 2′-deoxyguanosine and/or prodrug thereof and a PNP inhibitor. The PNP inhibitor has a Ki value of 50 nanomoles or less.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national phase of International ApplicationNo. PCT/US00/25306 filed Sep. 15, 2000, which claims priority from U.S.Provisional Patent Application Ser. No. 60/153,945 filed Sep. 15, 1999.

TECHNICAL FIELD

The present invention relates to enhancing the inhibition of T-cellproliferation in a mammalian host and especially a human host. Moreparticularly, the present invention is concerned with enhancing theinhibition of T-cell proliferation by administering 2′-deoxyguanosineand/or prodrugs thereof and certain PNP inhibitors which significantlyprolong the half-life of the 2′-deoxyguanosine in a host. The PNPinhibitors employed according to the present invention possess Ki valueof 50 nanomoles or less. The process of the present invention enhancesthe selective inhibition of T-cell proliferation without damaginghumoral immunity, which renders the process potentially effectiveagainst disorders in which T-cells are pathogenic.

BACKGROUND OF INVENTION

Purine nucleoside phosphorylase (PNP) deficiency is a rare inheriteddisease accounting for approximately 4% of patients with severe combinedimmunodeficiency. In PNP deficiency, T- and B-cell immunity areaffected. T-cell function may be profoundly deficient, may be normal atbirth and then decrease with time, or may fluctuate repeatedly betweenlow and normal. B-cell function can be normal but is deficient inapproximately one third of patients. PNP protein is a trimer ofapproximately 90,000 daltons. It is found in most tissues of the bodybut is at highest levels in lymphoid tissues. This tissue distributionexplains why the lymphoid system is predominantly affected in PNPdeficiency. Many mechanisms have been proposed to explain the metabolictoxicity in PNP deficiency. The elevated dGTP found in PNP deficiency isthought to inhibit ribonucleotide reductase and, thus, impede celldivision.

8-Aminoguanine given with 2′-deoxyguanosine inhibits the proliferationof human T-cells (CCRF-CEM and Molt-4 cells) in cultures.8-Aminoguanosine, a soluble derivative which is converted in vivo to8-aminoguanine, given to rats and dogs with 2′-deoxyguanosine causes aprofound fall in peripheral blood lymphocytes and was shown in rats toproduce increased levels of 2′-deoxyguanosine triphosphate (dGTP) inT-cells. To produce lymphopenia, inhibition of PNP was required, since2′-deoxyguanosine alone did not significantly decrease cell counts.

9-(3-Pyridylmethyl)-9-deazaguanine, one of a family of PNP inhibitors,increases plasma inosine levels in humans, indicating effectiveinhibition of PNP, but it did not reduce T-cell counts. The cell cultureand animal data presented above would indicate that in order to inhibitT-cell proliferation in humans, it would be necessary to provideexogenous 2′-deoxyguanosine, which would cause a sufficient accumulationof dGTP exclusively in the T-cells to inhibit their proliferation.However, exogenous 2′-deoxyguanosine rapidly degrades upon beingadministered to a host and therefore is not effective when administeredalone.

SUMMARY OF INVENTION

It has been found according to the present invention that administeringcertain PNP inhibitors in addition to the exogenous 2′-deoxyguanosineand/or prodrug of 2′-deoxyguanosine results in significantly prolongingthe half-life of the 2′-deoxyguanosine. Therefore, the combination of2′-deoxyguanosine and/or prodrug of 2′-deoxyguanosine and the PNPinhibitors employed according to the present invention provides apotentially effective treatment against disorders in which activatedT-cells are pathogenic. For instance, the implication of T-cells in thepathogenesis of various autoimmune diseases, including rheumatoidarthritis, systemic lupus erythematosus, psoriasis and type 1 diabetesstrongly suggests that the present invention will be an effectivetherapy for these diseases. Other indications are the prevention oforgan transplant rejection and the treatment of T-cell lymphomas andleukemias.

The PNP inhibitors employed according to the present invention have a Kivalue of 50 nanomoles or less. The PNP inhibitor can be administeredalong with or prior to the 2′-deoxyguanosine and/or prodrug of2′-deoxyguanosine.

Still other objects and advantages of the present invention will becomereadily apparent by those skilled in the art from the following detaileddescription, wherein it is shown and described only the preferredembodiments of the invention, simply by way of illustration of the bestmode contemplated of carrying out the invention. As will be realized theinvention is capable of other and different embodiments, and its severaldetails are capable of modifications in various obvious respects,without departing from the invention. Accordingly, the description is tobe regarded as illustrative in nature and not as restrictive.

Best and Various Modes for Carrying Out Invention

The present invention relates to enhancing the inhibition of T-cellproliferation in a mammalian host in need of such treatment andespecially a human host an effective amount of 2′-deoxyguanosine and/orprodrug of 2′-deoxyguanosine and an effective amount of at least one PNPinhibitor. The PNP inhibitor employed according to the present inventionhas a Ki value of 50 nanomoles or less.

Examples of suitable PNP inhibitors employed according to the presentinvention are those disclosed in U.S. Pat. Nos. 4,985,433; 4,985,434,5,008,265; 5,008,270; 5,565,463 and 5,721,240 assigned to BioCrystPharmaceuticals, Inc., disclosures of which are incorporated herein byreference. The preferred PNP inhibitor employed according to the presentinvention is 9-(3-pyridylmethyl)-9-deazaguanine.

Examples of suitable prodrugs of 2′-deoxyguanosine are represented bythe following:

wherein R₁ is Cl, NH₂, NHCH₃, R₃O, R₃S, or H;

R₂ is acyl typically having 1 to 6 carbon atoms, and

R₃ is alkyl typically having 1 to 3 carbon atoms and more typically 1carbon atom.

The first five structural types (i.e.—R₁ is Cl, NH₂, NHCH₃, R₃O or R₃S)are converted to 2′-deoxyguanosine in vivo by esterases and adenosinedeaminase.

The sixth type (R₁=H) is oxidized in vivo to 2′-deoxyguanosine. Examplesof these in vivo conversions are discussed in Montgomery, Prog. in Med.Chem. 7, 69 (1970) and Jones, Antiviral Chemistry and Chemotherapy 9,283 (1998). Mixtures of prodrugs can be employed, if desired, as well asmixtures of one or more prodrugs with 2′-deoxyguanosine.

According to the process of the present invention, the PNP inhibitor isadministered prior to or at the same time as the 2′-deoxyguanosineand/or prodrug of 2′-deoxyguanosine. The PNP should be present in thehost's bloodstream with 2′-deoxyguanosine in order to effectivelyprolong the half-life of the 2′-deoxyguanosine to permit a sufficientaccumulation of 2′-deoxyguanosine triphosphate in T-cells to preventtheir proliferation. When the PNP inhibitor is administered prior to the2′-deoxyguanosine and/or prodrug of 2′-deoxyguanosine it is typicallyadministered up to about 1 hour prior to the 2′-deoxyguanosine and/orprodrug of 2′-deoxyguanosine.

The compounds of the present invention can be administered by anyconventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, butgenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

The dosage administered will, of course, vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent and its mode and route of administration; the age, health andweight of the recipient; the nature and extent of the symptoms, the kindof concurrent treatment; the frequency of treatment; and the effectdesired. A daily dosage of active ingredient can be expected to be about0.001 to 1000 milligram (mg) per kilogram (kg) of body weight, with thepreferred dose being 0.1 to about 30 mg/kg.

Dosage forms (compositions suitable for administration) contain fromabout 1 mg to about 100 mg of active ingredient per unit. In thesepharmaceutical compositions, the active ingredient will ordinarily bepresent in an amount of about 0.5-95% by weight based on the totalweight of the composition.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, and powders, or in liquid dosage forms, suchas elixirs, syrups, and suspensions. It can also be administeredparenterally, in sterile liquid dosage forms. The active ingredient canalso be administered intranasally (nose drops) or by inhalation. Otherdosage forms are potentially possible such as administrationtransdermally, via a patch mechanism or ointment.

Gelatin capsules contain the active ingredient and powdered carriers,such as lactose, starch, cellulose derivatives, magnesium stearate,stearic acid, and the like. Similar diluents can be used to makecompressed tablets. Both tablets and capsules can be manufactured assustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can besugar-coated or film-coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration preferably contain awater-soluble salt of the active ingredient, suitable stabilizingagents, and, if necessary, buffer substances. Antioxidizing agents suchas sodium bisulfite, sodium sulfite, or ascorbic acid, either alone orcombined, are suitable stabilizing agents. Also used are citric acid andits salts and sodium EDTA. In addition, parenteral solutions can containpreservatives, such as benzalkonium chloride, methyl- or propylparaben,and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

Useful pharmaceutical dosage forms for administration of the compoundsaccording to the present invention can be illustrated as follows:

Capsules

A large number of unit capsules are prepared by filling standardtwo-piece hard gelatin capsules each with 100 mg of powdered activeingredient, 150 mg of lactose, 50 mg of cellulose, and 6 mg of magnesiumstearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil, or olive oil is prepared and injected by means of apositive displacement pump into gelatin to form soft gelatin capsulescontaining 100 mu of the active ingredient. The capsules are washed anddried.

Tablets

A large number of tablets are prepared by conventional procedures sothat the dosage unit was 100 mg of active ingredient, 0.2 mg ofcolloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg ofmicrocrystalline cellulose, 11 mg of starch, and 98.8 mg of lactose.Appropriate coatings may be applied to increase palatability or delayabsorption.

Moreover, the compounds of the present invention can be administered inthe form of nose drops or a nasal inhaler.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

The foregoing disclosure includes all the information deemed essentialto enable those skilled in the art to practice the claimed invention.Because the cited applications may provide further useful information,these cited materials are hereby incorporated by reference in theirentirety.

The foregoing description of the invention illustrates and describes thepresent invention. Additionally, the disclosure shows and describes onlythe preferred embodiments of the invention but, as mentioned above, itis to be understood that the invention is capable of use in variousother combinations, modifications, and environments and is capable ofchanges or modifications within the scope of the inventive concept asexpressed herein, commensurate with the above teachings and/or the skillor knowledge of the relevant art. The embodiments described hereinaboveare further intended to explain best modes known of practicing theinvention and to enable others skilled in the art to utilize theinvention in such, or other, embodiments and with the variousmodifications required by the particular applications or uses of theinvention. Accordingly, the description is not intended to limit theinvention to the form disclosed herein. Also, it is intended that theappended claims be construed to include alternative embodiments.

What is claimed is:
 1. A process for inhibiting T-cell proliferation in a mammalian host in need thereof by administering to said host an effective amount of at least one member selected from the group consisting of 2′-deoxyguanosine, prodrugs of 2′-deoxyguanosine and mixtures thereof, and at least one PNP inhibitor having a Ki of 50 nanomoles or less wherein said PNP inhibitor comprises 9-(3-pyridylmethyl)-9-deazaguanine.
 2. The process of claim 1 wherein the PNP inhibitor is administered simultaneously with the at least one member or prior to the at least one member.
 3. The process of claim 1 wherein the PNP inhibitor is administered up to about 1 hour prior to administering the at least one member.
 4. The process of claim 1 which comprises orally administering the inhibitor.
 5. The process of claim 4 which comprises administering the at least one member by infusion.
 6. The process of claim 4 which comprises orally administering the at least one member.
 7. The process of claim 1 wherein the at least one member is 2′-deoxyguanosine.
 8. The process of claim 5 wherein the at least one member is 2′-deoxyguanosine.
 9. The process of claim 6 wherein the at least one member is 2′-deoxyguanosine. 